SUMMARY: PROJECT 1 To improve the notoriously poor outcome of patients with malignant gliomas, we developed a novel oncolytic adenovirus, Delta-24-RGD, that selectively replicates in and destroys glioma cells. This virus was tested in a first-in-human Phase I clinical trial in patients with recurrent malignant gliomas (NCT00805376), in which dramatic complete (>95% tumor reduction) and durable (>3 years) responses were observed in 12% of patients. Data from this trial contributed to the groundbreaking paradigm shift demonstrating that the Delta-24-RGD oncolytic virus is a form of immunotherapy. Specifically, analyses of clinical responses and post treatment surgical specimens demonstrated that the oncolytic effect of Delta-24-RGD is followed by an anti-tumor cytotoxic T cell immune response that is capable of resulting in complete tumor regression in a small but significant percentage of patients. These clinical data emphasize the urgent need to amplify the anti-tumor immune response as a means of enhancing the efficacy of Delta-24-RGD. To this end, in this proposal we pursue two convergent approaches whose foundations rest on the concept that immune responses to tumors are mediated by 1) immune checkpoints molecules that attenuate immune responses and against which FDA-approved inhibitors are available, and 2) immune costimulatory molecules which activate immune responses and are ideal to ?arm? Delta-24-RGD. In our first approach, we combine Delta-24-RGD with the immune checkpoint inhibitor Pembrolizumab (MERCK), that is directed against the cell surface checkpoint receptor PD-1. We take advantage of the pretreatment biopsy specimens obtained from an ongoing Phase I/II clinical trial of this combination in patients with recurrent gliomas (the CAPTIVE trial, NCT02798406), to not only assess the safety and efficacy of this combination, but also to assess biomarkers for response (Aim 1). In our second approach, we develop and test next-generation Delta-24-RGD viruses that are armed with the cDNA of the ligands of immune co-stimulatory receptors (OX40L, GITRL, 4-1BB). We have already constructed and fully characterized the anti-glioma effects of Delta-24-RGDOX, which carries OX40L, and our data show that Delta-24-RGDOX more efficiently eradicates gliomas compared with Delta-24-RGD in immunocompetent animal models. Therefore, in Aim 2 of this proposal we assess the safety and biological effects of Delta-24-RGDOX on patient tumors in a unique treat-resect-treat clinical trial. Lastly, in Aim 3 we characterize the anti-glioma effects of additional next generation viruses Delta- 24-GREAT (which contains GITRL) and Delta-24-ACT (which contains 4-1BBL) alone and in combination with Delta-24-RGD, to define potential synergy of these viruses. If successful, Project 1 will usher in a new age of oncolytic viral therapies for the treatment of malignant gliomas, for which there is currently no effective treatment.